Commercial Protein Crystal Growth - High Density (CPCG-H) - 09.17.14

Science Objectives for EveryoneCommercial Protein Crystal Growth - High Density (CPCG-H) tested hardware using a variety of protein crystal growth methods. Researchers determined the type of hardware that would be most appropriate for each experiment and which type of hardware could be permanently added to or removed from ISS facilities for future protein crystal experiments. Protein crystal growth experiments aid the generation of computer models of carbohydrates, nucleic acids and proteins, and further advance the progress of biotechnology. Understanding these results will lead to advances in manufacturing and biological processes, both in medicine and agriculture.

The objective of the Commercial Protein Crystal Growth - High Density (CPCG-H) experiment was to produce large, well-ordered crystals of several different macromolecules for use in X-ray diffraction studies.

This investigation verified the performance of the new hardware assembly and continued to develop technology for macromolecular crystal growth experiments in microgravity.

The hardware consists of 4 independently operated trays each holding 42 growth cell assemblies of six experiments for a total of 1008 experiments. The growth cell assembly utilizes a user-friendly vapor diffusion chamber that mimics typical ground-based Linbro 24-well plates.

Description
The Commercial Protein Crystal Growth - High Density (CPCG-H) is protein crystal growth experiment flight hardware. During ISS Expeditions 2 and 4, CPCG-H was outfitted with High-Density Protein Crystal Growth (HDPCG) hardware. HDPCG was a vapor-diffusion facility that could process as many as 1008 individual protein samples. The entire HDPCG assembly had four independent trays that held 252 individual protein crystal growth experiments on each. The chambers had a protein reservoir, a precipitant reservoir, and an optically-clear access cap. The chambers were designed to reduce sedimentation problems and to produce highly uniform, single crystals. The trays can be removed and transferred to an awaiting camera system, Commercial Protein Crystal Growth - Video (CPCG-V), for observation while on the International Space Station (ISS). The individual experiments are grouped in sets of six and can be harvested one at a time.

The CPCG-H flight system can fly a typical Space Shuttle sortie mission or can be transferred to an ISS EXpedite the PRocessing of Experiments to Space Station (EXPRESS) Rack for an extended mission. The HDPCG growth cell assemblies can provide up to three levels of containment if needed for safety while providing in-process crystal observations through optically-clear polycarbonate windows. CPCG-H is a single Middeck Locker Equivalent which weighs 32.7 kg.

Proteins provide the building blocks of our bodies. Some proteins make it possible for red blood cells to carry oxygen while other proteins help transmit nerve impulses that allow us to see, hear, smell, and touch. Still other proteins play crucial roles in causing diseases. Pharmaceutical companies may be able to develop new or improved drugs to fight those diseases once the exact structures of the proteins are known.

The goal of the Commercial Protein Crystal Growth - High-density (CPCG-H) is to grow high-quality crystals of selected proteins so that their molecular structures can be studied. On Earth, gravity often has a negative impact on growing protein crystals. In microgravity, however, gravitational disturbances are removed, thus allowing some crystals to grow in a more regular and perfect form.

The primary proteins involved in the testing of the CPCG-H hardware during ISS Expeditions 2 and 4 were mistletoe lectin-I (ML-I), Thermus flavus 5S RNA, brefeldin A-ADP ribosylated substrate (BARS), and a triple mutant myoglobin (Mb-YQR). ML-I is a ribosome inactivating protein that can stop protein biosynthesis (creation of proteins) in cells, and is also a major component of drugs used in the treatment of cancer. Although the study of Thermus flavus 5S RNA has been ongoing for well over 30 years, the exact function of this protein remains obscure. Scientists believe that the crystallization of different domains of this protein may reveal functional properties. BARS is an enzyme involved in membrane fission, catalyzing the formation of phosphatidic acid by transfer. Mb-YQR was studied to assess the functional role of packing defects in proteins. The understanding of these protein structures will provide valuable insight into the role of these proteins for applications in the pharmaceutical industry.

Space Applications
The crystals grown in microgravity are able to grow larger and more organized than those grown on Earth. The results from this investigation may further human space exploration efforts by creating technological and biological advancements as a direct result from this research.

Earth Applications
This investigation is a validation of the Commercial Protein Crystal Growth-High Density (CPCG-H) facility. The CPCG-H will be used to grow large protein crystals of medical importance in an undisturbed, microgravity environment. High-Density crystals were grown to study the effectiveness of the CPCG-H in producing high-quality crystals that enhance postflight, Earth-based analysis.

Knowledge of precise three-dimensional molecular structure is a key component in biotechnology fields such as protein engineering and pharmacology. In order to obtain accurate data on the three-dimensional structure of protein crystals or other macromolecules, scientists employ a process called X-ray crystallography. Crystallographers construct computer models that reveal the complex structures of a protein molecule. In order to generate an accurate computer model, crystallographers must first crystallize the protein and analyze the resulting crystals by a process called X-ray diffraction. Precise measurements of thousands of diffracted intensities from each crystal help scientists map the probable positions of the atoms within each protein molecule. This complex process requires several months to several years to complete.

The quality of structural information obtained from X-ray diffraction methods is directly dependent on the degree of perfection of the crystals. Thus, the structures of many important proteins remain a mystery simply because researchers are unable to obtain crystals of high enough quality or large enough size. Generally, crystals must have dimensions of approximately 0.3 mm to 1.00 mm, and the protein molecules must be arranged in an orderly, repeating pattern. Consequently, the growth of high quality macromolecular crystals for diffraction analyses has been of primary importance for protein engineers, biochemists, and pharmacologists.

On Earth, the crystallization process is hindered by forces of sedimentation and convection since the molecules in the crystal solution are not of uniform size and weight. This leads to many crystals of irregular shape and small size that are unusable. However, the microgravity environment aboard the ISS is relatively free from the effects of sedimentation and convection and provides an exceptional environment for crystal growth.

Operational Requirements
The CPCG-H payload is a passive experiment and required minimal crew time. The hardware was equipped to provide data download on its health and status to the science team.

Operational Protocols
Samples were loaded into the hardware, preflight. The crew transferred the CPCG-H hardware between the Shuttle and its rack location. The hardware maintained and monitored the payload temperature and experiment status throughout the mission. Following transfer and activation via the crew interface controls on the front , crew involvement consisted of daily status checks, to make sure the hardware was operating nominally, and cleaning the filter once a week. Crystal samples grew throughout the duration of the mission and were returned to Earth for postflight analysis, such as X-ray crystallography and three-dimensional modeling.

Preliminary analysis indicated that at least 65% of the macromolecules flown in the CPCG-H experiments produced diffraction-sized crystals. X-ray diffraction studies of these crystals were conducted, and the data were used to determine and refine the three-dimensional structures of these macromolecules. Three benchmark proteins, ML-I, Thermus flavus 5S RNA, and BARS, were flown to validate the performance of the hardware. Diffraction-quality crystals, which were obtained from all of these proteins, yielded X-ray diffraction data comparable to those previously collected on Earth-grown crystals. Since the structure of each of the benchmark proteins is known to high resolution, these results indicate that the new HDPCG assembly worked very well, successfully producing high-quality crystals of the benchmark proteins.

Synchrotron diffraction data, collected from the space crystals of the BARS protein, were comparable in resolution but more intense and showed significantly less mosaicity than data from Earth-grown crystals. This indicates that the space-grown crystals had a higher order at the molecular level, and the X-ray diffraction data from the space crystals produced a more complete data set. These results contributed significantly to the structural study of BARS (Nardini et al. 2002).

ML-I is an enzyme that has the ability to inactivate ribosomes and inhibit cell replication. It is a target for new cancer treatments. Crystals of the protein attached to adenine (one of five building blocks of DNA or RNA) were flown, and these crystals yielded X-ray data to 1.9 angstrom. These data were used to refine the structure of the complex and were especially valuable in refining the active site conformation (Krauspenhaar et al. 2002).

Perhaps the most exciting results from the macromolecular crystallization experiments conducted in the CPCG-H hardware were obtained from the Thermus flavus 5S rRNA [ribosomal ribonucleic acid] experiments. These experiments involved a synthetic RNA duplex of 5S rRNA, which is a model system for the study of the binding of ribosomal RNA to proteins. Crystallization under microgravity provided crystals of significantly higher quality than those grown in one-g. The space crystals diffracted to a maximum resolution of 2.6 angstrom in contrast to the best Earth-grown crystals, which diffracted to 2.9 angstrom. The improved X-ray data facilitated the completion of the structure of the RNA segment (Vallazza et al. 2002).

To understand the true function of a protein, the structure must be determined. The model of the structure must be accurate to allow scientists to create compounds that bind to the protein. The understanding of the protein structure is of major importance with complex proteins (proteins that have significant folding). The three-dimensional structure of the triple mutant protein Mb-YQR was solved by growing the protein on ISS during Expeditions 2 and 4. Following return to Earth, three-dimensional models were created of the Mb-YQR proteins grown in space using X-ray crystallography techniques (Miele et al. 2004).

Structural studies of microgravity-grown crystals have provided important information for the development of new drugs. For example, previous studies conducted using crystals grown on shuttle flights have been used in the design of inhibitors, which may serve as broad-spectrum antibiotics. The CPCG-H payload offers a great increase in the amount of space available for protein crystal growth, enhancing the space station's research capabilities and commercial potential.